Bispecific OR-gate chimeric antigen receptor responsive to CD19 and CD20

ABSTRACT

A CD19-OR-CD20 chimeric antigen receptor (CAR) protein construct is provided. Also provided are nucleic acids encoding the CD19-OR-CD20 CAR; and methods of use, e.g. in the treatment of B cell malignancies. The CD19-OR-CD20 CAR of the invention is a bispecific CAR that can trigger T-cell activation upon detection of either CD19 or CD20 (or both). It is a single molecule that confers two-input recognition capability upon human T cells engineered to stably express this CAR.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371of International Application No. PCT/US2015/065620, filed Dec. 14, 2015,which claims the benefit of U.S. Provisional Application No. 62/091,854,filed Dec. 15, 2014, the contents of which applications are incorporatedinto the present application by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under OD012133, awardedby the National Institutes of Health. The Government has certain rightsin the invention. This work was supported by the U.S. Department ofVeterans Affairs, and the Federal Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Chimeric antigen receptors (CARs) are artificial molecules that redirectthe specificity of T cells to predetermined antigens. These receptorsare frequently used to graft the specificity of a monoclonal antibodyonto a T cell; with transfer of their coding sequence facilitated byretroviral or lentiviral vectors. Using adoptive transfer, autologous Tcells can be genetically modified ex vivo to express a CAR specific fora cancer cell of interest. The T cells, which can then recognize andkill the cancer cells, are reintroduced into the patient. Phase Iclinical studies of this approach have shown efficacy.

The most common form of CARs are fusions of single-chain variablefragments (scFv) derived from monoclonal antibodies, fused to CD3-zetasignaling domain, which contains 3 ITAMs. CD3-zeta may not provide afully competent activation signal and additional co-stimulatorysignaling is needed. For example, chimeric CD28 and OX40 can be usedwith CD3-Zeta to transmit a proliferative/survival signal, or all threecan be used together. Such molecules result in the transmission of azeta signal in response to recognition by the scFv of its target.

Multiple clinical trials have reported remarkable therapeutic efficacyof anti-CD19 CAR-modified T cells against both acute and chronic B-cellmalignancies. However, multiple cases have also been reported ofpatients relapsing with the emergence of CD19-negative leukemia orlymphoma (Maude et al. 2014). This problem of antigen escape, i.e.,tumor cells evading treatment by losing the antigen targeted by theT-cell therapeutic, is addressed by this invention.

SUMMARY OF THE INVENTION

CD20 and CD19 are both pan-B-cell markers present on the vast majorityof malignant B cells. An OR-gate CAR that triggers tumor killing as longas either CD20 or CD19 is present reduces the probability of antigenescape, by requiring that tumor cells lose both antigens to escapetargeting, an event that happens with a significantly lower probabilitythan single-antigen mutations. Therefore, this invention has a strongcompetitive advantage compared to the conventional, single-inputanti-CD19 CAR T-cell therapy.

A CD19-OR-CD20 chimeric antigen receptor (CAR) protein construct isprovided. Also provided are nucleic acids encoding the CD19-OR-CD20 CAR;and methods of use, e.g. in the treatment of B cell malignancies. TheCD19-OR-CD20 CAR of the invention is a bispecific CAR that can triggerT-cell activation upon detection of either CD19 or CD20 (or both). It isa single molecule that confers two-input recognition capability uponhuman T cells engineered to stably express this CAR. The CD19-OR-CD20CAR consists of the following (from N- to C-terminus): signal sequence;anti CD20 scFv; linker; anti-CD19 scFv; spacer domain; transmembranedomain; zero, one, or more cytoplasmic co-stimulatory signaling domains;CD3 zeta signaling domain. In some embodiments the spacer domain is animmunoglobulin hinge domain, including without limitation the human IgG4hinge.

In some embodiments the transmembrane domain is CD28 transmembranedomain. In some embodiments the cytoplasmic co-stimulatory signalingdomain is CD28 and/or 4-1BB. In some embodiment the construct furthercomprises T2A ribosomal skipping peptide, which can be used to link theCAR to a protein or peptide of interest, e.g. an epitope tag. In someembodiment a sortable tag is included, e.g. truncated epidermal growthfactor receptor (EGFRt) or fluorescent proteins, which can be used toseparate T cells expressing the CAR.

In some specific embodiments the linker joining the two scFv sequencesis a rigid linker. In some specific embodiments, a rigid linker has thesequence SEQ ID NO:1 (EAAAK)n, where n is 1, 2, 3, 4, 5, 6, etc. In somespecific embodiments, n is 3.

In some embodiments the CAR construct is packaged into a lentiviralvector, which includes, without limitation, a third-generationlentiviral vector. Primary human T cells can be lentivirally transducedto stably integrate and express the OR-gate CAR. CAR-expressing cellscan be enriched by fluorescence- or magnetism-activated cell sorting andexpanded by antigen stimulation or stimulation with CD3/CD28 antibodiesor antibody-coated microbeads.

In some embodiments of the invention, an expression vector encoding theCD19-or-CD20 CAR is provided, where the vector may be a lentiviralvector, a retroviral vector, an adenoviral vector, an adeno-associatedviral vector, a plasmid, or RNA.

In some embodiments, a method of killing a cancer cell in an individualis provided, comprising the step of providing to the individual atherapeutically effective amount of a therapeutic cell of the invention,including an effector cell, such as a T cell, NK cell, NKT cell, etc.,for example. The individual may have a B-cell malignancy, expressing onor both of CD20 and CD19. Any method of the invention may furthercomprise the step of delivering to the individual an additional cancertherapy, such as surgery, radiation, hormone therapy, chemotherapy,immunotherapy, or a combination thereof, for example.

In embodiments of the invention, a kit is provided comprising cellscomprising a CD19-or-CD20 CAR and/or expression vector encoding aCD19-or-CD20 CAR.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing.

FIG. 1. Schematic of bispecific, CD20-OR-CD19 CAR. The bispecific CAR iscomposed of (from N to C terminal): A signal sequence that directs CARlocalization to the cell membrane, the CD20 scFv, a peptide linker(e.g., (G4S)1, (G4S)3, SEQ ID NO:1 (EAAAK)1, or SEQ ID NO:1 (EAAAK)3),the CD19 scFv, followed by a spacer (e.g., the IgG4 hinge domain), atransmembrane domain (e.g., the transmembrane domain of CD28), one ormore co-stimulatory domains (e.g., the cytoplasmic domain of 4-1BB orCD28), and the cytoplasmic domain of CD3ζ chain. To facilitateidentification of CAR-expressing T cells by antibody staining, truncatedepidermal growth factor receptor (EGFRt) can be linked to the CAR via aself-cleaving peptide (e.g., T2A).

FIGS. 2A-2B. OR-gate CARs but not single-input CD19 CARs respond to Rajilymphoma cells that have undergone antigen escape. (A) CD69, CD137, andCD107a surface expression (in median fluorescence intensity; MFI) byCAR-T cells after a 24-hour co-incubation with CD19− Raji cells. (B)IFN-γ, TNF-α, and IL-2 production by the CAR-T cells in (A) as measuredby cytometric bead array assay. Mock: T cells that have been mocktransduced and do not express CARs. CD19 Short: single-input CD19 CARwith IgG4 hinge as spacer. CD20 Long: single-input CD20 CAR with IgG4hinge-CH2-CH3 as spacer. (G4S)1, (G4S)4, SEQ ID NO:1 (EAAAK)1, and SEQID NO:1 (EAAAK)3 indicate the linker sequence of CD20-OR-CD19 CARs, allof which contain the IgG4 hinge as spacer. Reported values are the meanof triplicates, with error bars indicating one standard deviation.P-values were calculated by two-tailed Student's t test; *: p<0.05; **:p<0.01.

FIG. 3. Cell lysis by single-input and OR-gate CAR-T cells after 4-hourco-incubation with wildtype (WT; CD19+/CD20+) or CD19− Raji(CD19−/CD20+) cells. Reported values are the mean of triplicates, witherror bars indicating one standard deviation. CAR identities are asdescribed in FIG. 2.

FIGS. 4A-4B. Bispecificity is not compromised CD19 detection by OR-gateCARs. CAR-T cells were co-incubated with wildtype Raji or CD19+ K562targets for 24 hours. (A) CD69, CD137, and CD107a surface expression wasquantified by flow cytometry. (B) IFN-γ, TNF-α, and IL-2 production wasquantified by cytometric bead array assay. Reported values are the meanof triplicates, with error bars indicating one standard deviation. CARidentities are as described in FIG. 2.

FIGS. 5A-5E. OR-gate CARs abrogate the effects of antigen escape invivo. (A) Tumor progression in NSG mice bearing wildtype (WT) or mixed(75% wildtype; 25% CD19−) Raji xenografts. Bioluminescence imaging wasperformed on days 6, 18, and 21 post tumor injection (T cells wereinjected on day 7). (B) Survival of mice bearing VVT or mixed Raji tumorxenografts and treated with T cells expressing no CAR or thesingle-input CD19 CAR. Results indicate single-input CD19 CAR is able tosignificantly extend the survival of animals engrafted with WT Rajitumors. (C) Survival of mice bearing WT Raji tumor xenografts andtreated with T cells expressing the single-input CD19 CAR or OR-gateCARs. Results indicate OR-gate CARs are as efficient as single-inputCD19 CAR in targeting wildtype Raji lymphoma. (D) Survival of micebearing mixed Raji tumor xenografts and treated with T cells expressingno CAR, the single-input CD19 CAR, or OR-gate CARs. Results indicateonly OR-gate CARs are able to significantly extend survival of animalsbearing CD19− mutant tumors. (E) Survival of mice bearing WT or mixedRaji tumor xenografts and treated with T cells expressing OR-gate CARs.Results indicate OR-gate CARs are equally efficient against WT and CD19−mutant Raji tumors, thus rendering the T cells insensitive to antigenloss by target cells. N=5 in all test groups. P-values were calculatedby log-rank test analysis; n.s.: not significant (p>0.1); *: p<0.1; **:p<0.05. CAR identities are as described in FIG. 2.

FIG. 6. Representative plasmid map of a bispecific, CD20-OR-CD19 CAR.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

The term “genetic modification” means any process that adds, deletes,alters, or disrupts an endogenous nucleotide sequence and includes, butis not limited to viral mediated gene transfer, liposome mediatedtransfer, transformation, transfection and transduction, e.g., viralmediated gene transfer such as the use of vectors based on DNA virusessuch as lentivirus, adenovirus, retroviruses, adeno-associated virus andherpes virus.

“Variant” refers to polypeptides having amino acid sequences that differto some extent from a native sequence polypeptide. Ordinarily, aminoacid sequence variants will possess at least about 80% sequenceidentity, more preferably, at least about 90% homologous by sequence.The amino acid sequence variants may possess substitutions, deletions,and/or insertions at certain positions within the reference amino acidsequence.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors, such as natural killer cells, neutrophils, andmacrophages, recognize bound antibody on a target cell and cause lysisof the target cell. ADCC activity may be assessed using methods, such asthose described in U.S. Pat. No. 5,821,337.

“Effector cells” are leukocytes which express one or more constantregion receptors and perform effector functions.

To “treat” a disease or a disorder, such as cancer, means to take eithertherapeutic measures or preventative measures to lessen or abate thedisease or disorder. Such treatment includes prevention, alleviation ofsymptoms, diminishment or stabilization of scope, and/or remission.

The term “therapeutically effective amount” refers to an amount of acompound or molecule effective to treat a disease or disorder.

“Cancer” refers to cells undergoing uncontrolled cellular growth.Examples of cancer include colorectal cancer and head and neck cancer. A“chemotherapeutic agent” is a chemical compound useful in the treatmentof cancer.

A “cytokine” is a protein released by one cell to act on another cell asan intercellular mediator.

“Non-immunogenic” refers to a material that does not initiate, provokeor enhance an immune response where the immune response includes theadaptive and/or innate immune responses.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region “leader and trailer” as well as intervening sequences(introns) between individual coding segments (exons). Some genes may bedeveloped which lack, in whole or in part, introns. Some leadersequences may enhance translation of the nucleic acid into polypeptides.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

As used herein, a “vector” may be any agent capable of delivering ormaintaining nucleic acid in a host cell, and includes viral vectors(e.g. retroviral vectors, lentiviral vectors, adenoviral vectors, oradeno-associated viral vectors), plasmids, naked nucleic acids, nucleicacids complexed with polypeptide or other molecules and nucleic acidsimmobilized onto solid phase particles. The appropriate DNA sequence maybe inserted into the vector by a variety of procedures. In general, theDNA sequence is inserted into an appropriate restriction endonucleasesite(s) by procedures known in the art. Such procedures and others aredeemed to be within the scope of those skilled in the art. Transcriptionof the DNA encoding the polypeptides of the present invention by highereukaryotes is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 by that act on a promoter to increase its transcription. Examplesincluding the SV40 enhancer on the late side of the replication originby 100 to 270, a cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers.

“Receptor” means a polypeptide that is capable of specific binding to amolecule. Whereas many receptors may typically operate on the surface ofa cell, some receptors may bind ligands when located inside the cell(and prior to transport to the surface) or may reside predominantlyintra-cellularly and bind ligand therein.

The term “antibody” includes monoclonal antibodies, polyclonalantibodies, dimers, multimers, multispecific antibodies and antibodyfragments that may be human, mouse, humanized, chimeric, or derived fromanother species. A “monoclonal antibody” is an antibody obtained from apopulation of substantially homogeneous antibodies that is beingdirected against a specific antigenic site.

“Antibody or functional fragment thereof” means an immunoglobulinmolecule that specifically binds to, or is immunologically reactive witha particular antigen or epitope, and includes both polyclonal andmonoclonal antibodies. The term antibody includes genetically engineeredor otherwise modified forms of immunoglobulins, such as intrabodies,peptibodies, chimeric antibodies, fully human antibodies, humanizedantibodies, and heteroconjugate antibodies (e.g., bispecific antibodies,diabodies, triabodies, and tetrabodies). The term functional antibodyfragment includes antigen binding fragments of antibodies, includinge.g., Fab′, F(ab′)₂, Fab, Fv, rlgG, and scFv fragments. The term scFvrefers to a single chain Fv antibody in which the variable domains ofthe heavy chain and of the light chain of a traditional two chainantibody have been joined to form one chain.

The use of a single chain variable fragment (scFv) is of particularinterest. scFvs are recombinant molecules in which the variable regionsof light and heavy immunoglobulin chains encoding antigen-bindingdomains are engineered into a single polypeptide. Generally, the V_(H)and V_(L) sequences are joined by a linker sequence. See, for example,Ahmad (2012) Clinical and Developmental Immunology Article ID 980250,herein specifically incorporated by reference.

The length of the DNA linker used to link both of the domains isimportant for proper folding. It has been estimated that the peptidelinker must span 3.5 nm (35 Å) between the carboxy terminus of thevariable domain and the amino terminus of the other domain withoutaffecting the ability of the domains to fold and form an intactantigen-binding site. Many such linkers are known in the art, forexample flexible linkers comprising stretches of Gly and Ser residues.The linkers used in the present invention include, without limitation, arigid linker. In some specific embodiments of the invention, a rigidlinker has the sequence SEQ ID NO:1 (EAAAK)n, where n is 1, 2, 3, 4, 5,6, etc. In some specific embodiments, n is 3.

Spacer. A spacer region links the antigen binding domain to thetransmembrane domain. It should be flexible enough to allow the antigenbinding domain to orient in different directions to facilitate antigenrecognition. The simplest form is the hinge region from animmunoglobulin, e.g. the hinge from any one of IgG1, IgG2a, IgG2b, IgG3,IgG4, particularly the human protein sequences. Alternatives include theCH₂CH₃ region of immunoglobulin and portions of CD3. For many scFv basedconstructs, an IgG hinge is effective.

T2A peptide. T2A peptide can be used to link the CAR of the invention toan epitope tag or other protein or peptide, including without limitationa sortable tag. T2A-linked multicistronic vectors can be used to expressmultiple proteins from a single open reading frame. The small T2Apeptide sequences, when cloned between genes, allow for efficient,stoichiometric production of discrete protein products within a singlevector through a novel “cleavage” event within the T2A peptide sequence.Various 2A peptide sequences are known and used in the art, for examplesee Szymczak-Workman et al. (2012) Cold Spring Harb Protoc.2012(2):199-204, herein specifically incorporated by reference. They aresmall (18-22 amino acids) and have divergent amino-terminal sequences,which minimizes the chance for homologous recombination and allows formultiple, different 2A peptide sequences to be used within a singlevector.

As used herein, the term “tumor microenvironment” refers to any and allelements of the tumor milieu that creates a structural and or functionalenvironment for the malignant process to survive and/or expand and/orspread.

CD20 is a cell surface protein present on most B-cell neoplasms, andabsent on otherwise similar appearing T-cell neoplasms. CD20 positivecells are also sometimes found in cases of Hodgkins disease, myeloma,and thymoma. CD20 is the target of the monoclonal antibodies (mAb)rituximab, ofatumumab, ocrelizumab, genmab, obinutuzumab, Ibritumomabtiuxetan, AME-133v, IMMU-106, TRU-015, and tositumomab, which are allactive agents in the treatment of all B cell lymphomas and leukemias.For the purposes of the present invention, any of these antibodies maybe converted into a scFv and used in the CAR. In some embodiments, thescFv is derived from Leu16 monoclonal antibody.

Cancers that may be treated with anti-CD20 reagents, e.g. antibodies andCARs, include without limitation B-cell lymphomas and leukemias, forexample B-cell non-Hodgkin lymphomas (NHL), e.g. follicular lymphoma;hairy cell leukemia, and B-cell chronic lymphocytic leukemia (CLL).Anti-CD20 reagents are also useful in treating melanoma, e.g. targetingmelanoma cancer stem cells.

CD19 expression is a hallmark of B cells. CD19 antigen is a type Itransmembrane glycoprotein belonging to the immunoglobulin Igsuperfamily. CD19 is specifically expressed in normal B cells andneoplastic B cells. It is considered a pan B-cell marker expressedthroughout B-cell development but with threefold higher expression inmature cells as compared to immature B cells. CD19 expression however,is lost in the terminally differentiated plasma cells. Duringlymphopoiesis, CD19 directs B-cell fate and differentiation bymodulating B-cell receptor signaling. It is critically involved inestablishing the optimal immune response through its roles in theantigen-independent development as well as the immunoglobulin-inducedactivation of B cells. CD19 deficiency in humans and mice leads to anoverall impaired humoral response with increased susceptibility toinfection.

The pattern of CD19 expression is maintained among B-cell malignancieswhere it is expressed in indolent and aggressive subtypes of B celllymphomas and leukemias, including NHL, B-cell CLL, and non-T acutelymphoblastic leukemia (ALL). CD19 is expressed in the B-cell lineage atan earlier stage compared with CD20. This fact therefore, may provide anadvantage to CD19 targeted drugs over rituximab, especially for earlyB-cell neoplasms like acute lymphoblastic leukemia. Moreover, CD19 isshown to be internalized efficiently in lymphoma tumor models with theuse of different monoclonal antibodies (huB4, hBU12). Various anti-CD19antibodies can be formatted for use in the constructs of the presentinvention, including without limitation huB4, which is a humanizedanti-CD19 antibody. In some embodiments of the invention, the anti-CD19scFv is the FMC63 antibody.

Highly selective targeted T cell therapies are emerging as effectivenon-toxic modalities for the treatment of cancer. Malignancies arecomplex diseases where multiple elements contribute to the overallpathogenesis through both distinct and redundant mechanisms. Hence,targeting different cancer-specific markers simultaneously could resultin better therapeutic efficacy. However, developing two separatecellular products for clinical use as combination therapy isimpractical, owing to regulatory hurdles and cost. In contrast,rendering an individual T cell bispecific offsets tumor escape becauseof antigen loss.

In one embodiment, the bispecific CAR comprises a modified endogenouscell-surface molecule that may be used as a non-immunogenic selectionepitope compatible with immunomagnetic selection. Non-immunogenicepitopes are those that normally do not raise an immune response inhumans, and are usually proteins normally expressed in humans, orfragments thereof. Such a non-immunogenic selection epitope mayfacilitate immunotherapy in cancer patients without undesirableimmunologic rejection of cell products. The endogenous cell surfacemolecule may be modified or truncated to retain an extracellular epitoperecognized by a known antibody or functional fragment thereof, and toremove any signaling or trafficking domains and/or any extracellulardomains unrecognized by said known antibody. A modified endogenous cellsurface molecule which lacks a signaling or trafficking domain and/orany extracellular domains unrecognized by said known antibody isrendered inert. In some embodiments a truncated EGFR is used for thispurpose.

The modified endogenous cell-surface molecule may be, but is not limitedto, any non-immunogenic cell-surface related receptor, glycoprotein,cell adhesion molecule, antigen, integrin or cluster of differentiation(CD) that is modified as described herein. Modification of suchcell-surface molecules is accomplished by keeping an epitope that isrecognized by a known antibody or functional fragment thereof; andremoving any signaling or trafficking domains and/or any extracellulardomains unrecognized by a known antibody. Removal of the signaling ortrafficking domains and/or any extracellular domains unrecognized by aknown antibody renders the endogenous cell-surface moleculenon-immunogenic and/or inert.

Thus, embodiments of the invention utilize an OR gate CAR as anartificial molecule that enables immune cells (T cells) to specificallyand distinctly recognize and attack two cancer target moleculessimultaneously, or to attack a cancer cell that has lost expression ofeither CD20 or CD19. The CAR is an artificial molecule that can begrafted onto T cells using genetic engineering technology to render themspecific to a target of interest. This ability has substantialtherapeutic implications, in that escape from single activity CARs hasbeen reported.

The CAR architecture may be any suitable architecture, as known in theart. In certain embodiments, a cytoplasmic signaling domain, such asthose derived from the T cell receptor ζ-chain, is employed as at leastpart of the chimeric receptor in order to produce stimulatory signalsfor T lymphocyte proliferation and effector function followingengagement of the chimeric receptor with the target antigen. Exampleswould include, but are not limited to, endodomains from co-stimulatorymolecules such as CD28, 4-1BB, and OX40 or the signaling components ofcytokine receptors such as IL7 and IL15. In particular embodiments,co-stimulatory molecules are employed to enhance the activation,proliferation, and cytotoxicity of T cells produced by the CAR afterantigen engagement. In specific embodiments, the co-stimulatorymolecules are CD28, OX40, and 4-1BB and cytokine and the cytokinereceptors are IL7 and IL15. The CAR may be first generation, secondgeneration, or third generation CAR, in which signaling is provided byCD3ζ together with co-stimulation provided by CD28 and a tumor necrosisfactor receptor (TNFr), such as 4-1BB or OX40), for example.

Embodiments of the invention include cells that express an OR-gate CARof the invention. The cell may be of any kind, including an immune cellcapable of expressing the OR-gate CAR of the invention for cancertherapy or a cell, such as a bacterial cell, that harbors an expressionvector that encodes the OR-gate CAR of the invention. As used herein,the terms “cell,” “cell line,” and “cell culture” may be usedinterchangeably. All of these terms also include their progeny, which isany and all subsequent generations. It is understood that all progenymay not be identical due to deliberate or inadvertent mutations. In thecontext of expressing a heterologous nucleic acid sequence, “host cell”refers to a eukaryotic cell that is capable of replicating a vectorand/or expressing a heterologous gene encoded by a vector. A host cellcan, and has been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid. In embodiments of the invention, a host cell is a T cell,including a cytotoxic T cell (also known as TC, Cytotoxic T Lymphocyte,CTL, T-Killer cell, cytolytic T cell, CD8+ T cells or killer T cell); NKcells and NKT cells are also encompassed in the invention.

The cells can be autologous cells, syngeneic cells, allogeneic cells andeven in some cases, xenogeneic cells. In many situations one may wish tobe able to kill the modified CTLs, where one wishes to terminate thetreatment, the cells become neoplastic, in research where the absence ofthe cells after their presence is of interest, or other event. For thispurpose one can provide for the expression of certain gene products inwhich one can kill the modified cells under controlled conditions, suchas inducible suicide genes.

By way of illustration, cancer patients or patients susceptible tocancer or suspected of having cancer may be treated as follows. Cancersinclude particularly B-cell leukemias and lymphomas. CTLs modified asdescribed herein may be administered to the patient and retained forextended periods of time. The individual may receive one or moreadministrations of the cells. In some embodiments, the geneticallymodified cells are encapsulated to inhibit immune recognition and placedat the site of the tumor. The cells may be injected at the tumor site orinjected intravenously, for example.

In particular cases the individual is provided with therapeutic CTLsmodified to comprise an OR-gate CAR of the invention. The cells may bedelivered at the same time or at different times as another type ofcancer therapy. The cells may be delivered in the same or separateformulations as another type of cancer therapy. The cells may beprovided to the individual in separate delivery routes as another typeof cancer therapy. The cells may be delivered by injection at a tumorsite or intravenously or orally, for example. Routine delivery routesfor such compositions are known in the art.

Expression vectors that encode the OR-gate CAR of the invention can beintroduced as one or more DNA molecules or constructs, where there maybe at least one marker that will allow for selection of host cells thatcontain the construct(s). The constructs can be prepared in conventionalways, where the genes and regulatory regions may be isolated, asappropriate, ligated, cloned in an appropriate cloning host, analyzed byrestriction or sequencing, or other convenient means. Particularly,using PCR, individual fragments including all or portions of afunctional unit may be isolated, where one or more mutations may beintroduced using “primer repair”, ligation, in vitro mutagenesis, etc.,as appropriate. The construct(s) once completed and demonstrated to havethe appropriate sequences may then be introduced into the CTL by anyconvenient means. The constructs may be integrated and packaged intonon-replicating, defective viral genomes like Adenovirus,Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others,including retroviral vectors or lentiviral vectors, for infection ortransduction into cells. The constructs may include viral sequences fortransfection, if desired. Alternatively, the construct may be introducedby fusion, electroporation, biolistics, transfection, lipofection, orthe like. The host cells may be grown and expanded in culture beforeintroduction of the construct(s), followed by the appropriate treatmentfor introduction of the construct(s) and integration of theconstruct(s). The cells are then expanded and screened by virtue of amarker present in the construct. Various markers that may be usedsuccessfully include hprt, neomycin resistance, thymidine kinase,hygromycin resistance, etc.

In some instances, one may have a target site for homologousrecombination, where it is desired that a construct be integrated at aparticular locus. For example, one can knock-out an endogenous gene andreplace it (at the same locus or elsewhere) with the gene encoded for bythe construct using materials and methods as are known in the art forhomologous recombination. For homologous recombination, one may useeither omega or O-vectors. Vectors containing useful elements such asbacterial or yeast origins of replication, selectable and/or amplifiablemarkers, promoter/enhancer elements for expression in prokaryotes oreukaryotes, etc. that may be used to prepare stocks of construct DNAsand for carrying out transfections are well known in the art, and manyare commercially available.

The CTLs that have been modified with the construct(s) are then grown inculture under selective conditions and cells that are selected as havingthe construct may then be expanded and further analyzed, using, forexample; the polymerase chain reaction for determining the presence ofthe construct in the host cells. Once the modified host cells have beenidentified, they may then be used as planned, e.g. expanded in cultureor introduced into a host organism.

Depending upon the nature of the cells, the cells may be introduced intoa host organism, e.g. a mammal, including humans, in a wide variety ofways. The cells may be introduced at the site of the tumor, in specificembodiments, although in alternative embodiments the cells hone to thecancer or are modified to hone to the cancer. The number of cells thatare employed will depend upon a number of circumstances, the purpose forthe introduction, the lifetime of the cells, the protocol to be used,for example, the number of administrations, the ability of the cells tomultiply, the stability of the recombinant construct, and the like. Thecells may be applied as a dispersion, generally being injected at ornear the site of interest. The cells may be in aphysiologically-acceptable medium.

The cells may be administered as desired. Depending upon the responsedesired, the manner of administration, the life of the cells, the numberof cells present, various protocols may be employed. The number ofadministrations will depend upon the factors described above at least inpart.

In some embodiments AAV, retroviral or lentiviral vectors are used todeliver the OR-gate CAR of the invention to a T cell.

Adeno associated virus (AAV) is an attractive vector system for use inthe cells of the present invention as it has a high frequency ofintegration and it can infect nondividing cells, thus making it usefulfor delivery of genes into mammalian cells, for example, in tissueculture or in vivo. AAV has a broad host range for infectivity. Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein byreference.

Retroviruses are useful as delivery vectors because of their ability tointegrate their genes into the host genome, transferring a large amountof foreign genetic material, infecting a broad spectrum of species andcell types and of being packaged in special cell lines.

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art.Some examples of lentivirus include the Human Immunodeficiency Viruses:HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Recombinantlentiviral vectors are capable of infecting non-dividing cells and canbe used for both in vivo and ex vivo gene transfer and expression ofnucleic acid sequences. In some embodiments the lentiviral vector is athird generation vector (see, for example, Dull et al. (1998) J Virol.72 (11):8463-71). Such vectors are commercially available. 2ndgeneration lentiviral plasmids utilize the viral LTR promoter for geneexpression, whereas 3rd-generation transfer vectors utilize a hybrid LTRpromoter, see, for example Addgene for suitable vectors.

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, one or more cells for use in cell therapy and/orthe reagents to generate one or more cells for use in cell therapy thatharbors recombinant expression vectors may be comprised in a kit. Thekit components are provided in suitable container means. Some componentsof the kits may be packaged either in aqueous media or in lyophilizedform. The container means of the kits will generally include at leastone vial, test tube, flask, bottle, syringe or other container means,into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the components in close confinement for commercial sale.Such containers may include injection or blow molded plastic containersinto which the desired vials are retained.

In certain embodiments of the invention, methods of the presentinvention for clinical aspects are combined with other agents effectivein the treatment of hyperproliferative disease, such as anti-canceragents. An “anti-cancer” agent is capable of negatively affecting cancerin a subject, for example, by killing cancer cells, inducing apoptosisin cancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cancer cells withthe expression construct and the agent(s) or multiple factor(s) at thesame time. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second agent(s).

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, abraxane, altretamine, docetaxel, herceptin,methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing and alsocombinations thereof.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

All references cited in this specification are hereby incorporated byreference in their entirety. The following examples are solely for thepurpose of illustrating one embodiment of the invention.

EXPERIMENTAL Bispecific CD20-OR-CD19 CAR

A bispecific CAR was constructed to have, from N to C terminal, a signalsequence that directs CAR localization to the cell membrane, the CD20scFv, a peptide linker, the CD19 scFv, followed by a spacer (e.g., theIgG4 hinge domain), a transmembrane domain (e.g., the transmembranedomain of CD28), none or one or more co-stimulatory domains (e.g., thecytoplasmic domain of 4-1BB or CD28), and the cytoplasmic domain of CD3ζchain. To facilitate identification of CAR-expressing T cells byantibody staining, truncated epidermal growth factor receptor (EGFRt)can be linked to the CAR via a self-cleaving peptide (e.g., T2A).

The amino acid sequence of various components is as follows:

GMpCSF signal sequence, (SEQ ID NO: 2) METDTLLLWVLLLWVPGSTG CD20 scFv(SEQ ID NO: 3) DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS CD19 scFv (SEQ ID NO: 4)DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS IgG4 Hinge (SEQ ID NO: 5)ESKYGPPCPPCP CD28 transmembrane domain (SEQ ID NO: 6)MFWVLVVVGGVLACYSLLVTVAFIIFWV CD28 cytoplasmic domain (SEQ ID NO: 7)RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 4-1BB cytoplasmic domain(SEQ OD NO: 8) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3ζcytoplasmic domain (SEQ ID NO: 9)RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRT2A self-cleaving peptide (SEQ ID NO: 10) LEGGGEGRGSLLTCGDVEENPGPRIgG4 CH2 (SEQ ID NO: 11)APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAK IgG4 CH3(SEQ ID NO: 12) GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGKTruncated epidermal growth factor receptor (EGFRt) (SEQ ID NO: 13)MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVA LGIGLFM

OR-gate CARs but not single-input CD19 CARs respond to Raji lymphomacells that have undergone antigen escape, as shown in FIG. 2 byexpression of CD69, CD137 and CD1-7a on the surface of theCAR-expressing T cells, and by the release of cytokines. The controlsinclude a CD19 Short single-input CD19 CAR with IgG4 hinge as spacer;and CD20 Long single-input CD20 CAR with IgG4 hinge-CH2-CH3 as spacer.Various linkers were tested, including (G4S)1, (G4S)4, (SEQ ID NO:1,EAAAK)1, and (D NO:1, EAAAAK)3.

A comparison of cell lysis by single-input and OR-gate CAR-T cells after4-hour co-incubation with wildtype (WT; CD19+/CD20+) or CD19− Raji(CD19−/CD20+) cells is shown in FIG. 3. The bispecificity was notcompromised in OR-gate CARs. CAR-T cells were co-incubated with VVT Rajior CD19+ K562 targets for 24 hours, and the expression of relevantactivation-induced antigens and release of cytokines are shown in FIG.4.

In vivo data, shown in FIG. 5, show OR-gate CARs abrogate the effects ofantigen escape. In the survival of mice bearing WT or mixed Raji tumorxenografts and treated with T cells expressing no CAR or thesingle-input CD19 CAR, the results showed that single-input CD19 CAR wasable to significantly extend the survival of animals engrafted with VVTRaji tumors, and that OR-gate CARs are as efficient as single-input CD19CAR in targeting WT Raji lymphoma. However, only OR-gate CARs were ableto significantly extend survival of animals bearing CD19− mutant tumors.OR-gate CARs are equally efficient against VVT and CD19− mutant Rajitumors, thus rendering the T cells insensitive to antigen loss by targetcells.

What is claimed is:
 1. A method of killing tumor cells in a cancerpatient comprising administering to the patient a population of T cellsexpressing a polypeptide comprising a CD19-O R-CD20 chimeric antigenreceptor (CAR) comprising, in order: anti-CD20 scFv comprising a lightchain variable region and heavy chain variable region from SEQ ID NO:3;(G45)4 linker; anti-CD19 scFv comprising a heavy chain variable regionand light chain variable region from SEQ ID NO:4; spacer domain of SEQID NO:5; transmembrane domain of SEQ ID NO:6; 4-1BB cytoplasmicsignaling domain of SEQ ID NO:8; and CD3 zeta cytoplasmic signalingdomain of SEQ ID NO:9, wherein the tumor cells express CD19 or CD20. 2.The method of claim 1, wherein the tumor cells are lymphoma cells. 3.The method of claim 1, wherein the transmembrane domain is CD28transmembrane domain.
 4. The method of claim 1, wherein the CAR furthercomprises sortable tag.
 5. The method of claim 4, wherein the sortabletag is truncated EGFR.
 6. The method of claim 4, wherein the sortabletag is a fluorescent protein.
 7. The method of claim 1, wherein thepopulation of cells abrogates antigen loss by the tumor cells.
 8. Amethod of treating a B-cell leukemia or lymphoma in a patient comprisingadministering to the patient a population of T cells expressing apolypeptide comprising a CD19-0R-CD20 chimeric antigen receptor (CAR)comprising, in order: anti-CD20 scFv comprising a light chain variableregion and heavy chain variable region from SEQ ID NO:3; (G45)4 linker;anti-CD19 scFv comprising a heavy chain variable region and light chainvariable region from SEQ ID NO:4; spacer domain of SEQ ID NO:5;transmembrane domain of SEQ ID NO:6; 1BB cytoplasmic signaling domain ofSEQ ID NO:8; and CD3 zeta cytoplasmic signaling domain of SEQ ID NO:9,wherein the B-cell leukemia or lymphoma expresses CD19 or CD20.
 9. Themethod of claim 8, wherein the population of cells abrogates antigenloss by the cancer cells.
 10. The method of claim 1, wherein the patientis a human patient.
 11. The method of claim 8, wherein the patient is ahuman patient.